Visible photorelease of liquid biopsy markers following microfluidic affinity-enrichment

Article abstract of DOI:10.1039/c9cc09598e

We detail a heterobifunctional, 7-aminocoumarin photocleavable (PC) linker with unique properties to covalently attach Abs to surfaces and subsequently release them with visible light (400-450 nm). The PC linker allowed rapid (2 min) and efficient (>90%) release of CTCs and EVs without damaging their molecular cargo.

Full text of DOI:10.1039/c9cc09598e

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Pahattuge, M. Jackson, R. digamber, H. Wijerathne, V. Brown, M. A. Witek, C. Perera, R. S. Givens and B.
Peterson, Chem. Commun., 2020, DOI: 10.1039/C9CC09598E.
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DOI: 10.1039/C9CC09598E
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Visible Photorelease of Liquid Biopsy Markers
following Microfluidic Affinity-Enrichment
Thilanga N. Pahattuge,†[a] J. Matt Jackson,†[a] Digamber Rane,[b] Harshani Wijerathne,[a] Virginia
Brown,[c] Malgorzata A. Witek,[a] Chamani Perera,[b] Richard S. Givens,[a] Blake R. Peterson,[b] and
Steven A. Soper*[a,c,d,e]
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
expression analyses.10 Thus, it is necessary to release affinity-
enriched markers without damaging the marker or its cargo.
We detail a heterobifunctional, 7-aminocoumarin photocleavable
(PC) linker with unique properties to covalently attach Abs to
surfaces and subsequently release them with visible light (400-450
nm). The PC linker allowed rapid (2 min) and efficient (>90%)
release of CTCs and EVs without damaging their molecular cargo.
New chemistries have been developed for “catch and release,”
where biomarkers are affinity-enriched on a solid-phase (i.e.,
microfluidic device) then released for analyses.11, 12 Release has been
accomplished using degraded polymer coatings, proteolytically
digested Abs, or cleaved Ab linkers with enzymes or UV light.2
Performance metrics for CTC catch and release are: (i) Recovery (CTC
yield/input); (ii) purity (CTCs/total cell count); (iii) release efficiency
(CTCs released/recovered); (iv) cell viability; (v) preserving molecular
cargo; and (vi) minimizing assay cost, workflow, and instrumentation.
Previously, we reported a single-stranded oligonucleotide linker
for Ab immobilization and subsequent enzymatic release. Enzymatic
cleavage of a dU nucleotide in the linker released >90% of CTCs,
preserved viability (>85%), and enabled off-line immunophenotyping
and cytogenetic analyses.13 We incorporated the oligonucleotide
linker into a sinusoidal microfluidic chip that achieved >75% CTC
Liquid biopsies consist of disease-associated markers harvested from
body fluids that can be secured in a minimally invasive manner to
provide information for guiding patient treatment (i.e., precision
medicine) by securing molecular characteristics of the disease.1
Initially focused on epithelial cancers, liquid biopsies have been
extended to other diseases such as blood cancers and stroke.2, 3
Common liquid biopsy markers include, but are not limited to,
nanometer-sized extracellular vesicles, EVs,4 and cells (circulating
tumour cells, CTCs,2 or CD8(+) T-cells for stroke5).
Microfluidics that use affinity-agents (e.g., Abs) attached to their
surfaces can specifically enrich disease-associated EVs1 or CTCs2 from
biological samples. Further, affinity-enrichment can fractionate
different marker subsets, such as targeting epithelial CTCs via EpCAM
(epithelial cell adhesion molecule) and mesenchymal CTCs via FAPα
(fibroblast activation protein alpha).6
recovery from clinical samples, high purity (>90%)13 and 80-100% test
6
positivity for several epithelial cancers.2,
We also used the
oligonucleotide linker to enrich leukemic cells3 and immune cells
responding to inflammatory processes associated with acute
ischemic stroke (AIS).5 However, the reaction time (~60 min) was
prohibitively long for time-sensitive analyses as required for AIS
diagnosis, where the therapeutic time window for recombinant
tissue plasminogen activator (r-tPA) treatment is ~4.5 h.5
While early studies focused on biomarker enumeration to
indicate disease status, the Precision Medicine initiative now
requires profiling the disease’s molecular composition.7 Thus, there
is a need to integrate enrichment with advanced molecular profiling.2,
We report a photocleavable 7-amino coumarin Ab linker for
specific biomarker enrichment and reagent-less release (catch and
release). 7- amino coumarin was used previously as a photolabile
group to release different targets (drugs,14, 15 thiol bearing proteins16
and CTCs17) over a wide wavelength range (UV, visible, and near-IR),
but UV induced DNA/RNA damage, low release efficiencies (50-80%)
and lengthy release times (~120 min) limited their use. Here, we
5,
8
For nanometer-sized EVs, enumeration requires off-line
nanoparticle tracking analysis (NTA) and transmission electron
microscopy (TEM) for enumeration,9 data critical for mRNA
[a]
T.N. Pahattuge, Dr. J.M. Jackson, H. Wijerathne, Dr. M.A. Witek, Prof.
R.S. Givens, Prof. S.A. Soper, Center of BioModular Multi-Scale Systems,
Department of Chemistry, University of Kansas, 1567 Irving Hill Rd.,
Lawrence, KS 66045; E-mail: ssoper@ku.edu
synthesized
a 7-(diethylamino)coumaryl-4-methyl photorelease
agent18 (see Scheme S1 and SI for synthesis of the PC linker and
structural characterization) that rapidly cleaves (Figure S1A) with
visible light (400–450 nm,19 quantum efficiency of coumarin dye is
0.2518) minimizing nucleic acid damage and little to no side reactions
(Scheme 1). Also, minimal changes in the UV/vis (Figure S1B) and
fluorescence emission (Figure S1C) as a function of cleavage were
observed. The PC linker is unique in its structure; the PC linker
contains amino and carboxy termini (i.e., amino acid) to allow for two
well-established EDC/NHS reactions to first covalently attach the
linker to a carboxylated surface, and then to an Ab.
†
These authors contributed equally to this work.
[b]
Dr. D. Rane, Dr. C. Perera, Prof. B.R. Peterson, Synthetic Chemical
Biology Core Laboratory, Department of Medicinal Chemistry, University
of Kansas, 2034 Becker Dr, Lawrence, KS 66047
[c]
[d]
[e]
V. Brown, Prof. S.A. Soper, Department of BioEngineering University of
Kansas, 1530 West 15th St., Lawrence, KS 66045
Department of Mechanical Engineering
3138 Learned Hall, 1530 West 15th St., Lawrence, KS 66045
Department of Cancer Biology and KU Cancer Center
University of Kansas Medical Center, Kansas City, KS
Supporting information for this article is given via a link at the end of the
document.
We will demonstrate efficient and rapid release with inexpensive
LEDs with minimal effects on the marker and its molecular cargo.
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was used with dry ACN to facilitate amide bond formation between
Also, we show that the PC linker can be used for the catch and release
of CTCs and EVs with no to minimal damage to their molecular cargo.
View Article Online
the primary amine and NHS ester. Unreacted NHS ester was
DOI: 10.1039/C9CC09598E
quenched with Tris base to prevent direct attachment of Cy5-labeled
oligonucleotides. Then, we reacted the linker’s -COOH terminus with
EDC/NHS in ACN, and immobilized Cy5-labeled oligonucelotides in
buffer using the PC linker (Figure 1A). We then used an LED (λmax
=
412 nm, 32 ±4 mW/cm2, Figure S3) to cleave the PC linker.
Decreasing on-chip fluorescence of the Cy5-labeled oligonucleotide
(Figure 1A and 1B) was confirmatory for successful release through
controls (Figure S4) and quantification of Cy5-labeled
oligonucleotides in the off-chip effluent by fluorescence
spectroscopy (Figure 1C). The remaining on-chip fluorescence may
be due to autofluorescence from the UV/O3 activated surface and/or
non-released Cy5-labeled oligonucleotides.
Scheme 1. Structure of the heterobifunctional PC linker and immobilization strategy
employing two EDC/NHS coupling reactions. The PC linker is immobilized to
carboxylated (UV/O3-oxidized) surfaces via the linker’s primary amine group. Abs are
then anchored to the linker’s -COOH handle. After biomarker purification, a visible
LED (400-450 nm) cleaves the PC linker. Immobilization reactions are conducted in
organic solvent to mitigate NHS ester hydrolysis, which could lead to direct (and non-
releasable) Ab conjugation to the -COOH surface.
The immobilization chemistry was designed to allow for the facile
surface attachment of a recognition element, for example an Ab,
used in a microfluidic device, which in this case was fabricated in
cyclic olefin copolymer (COC) for enriching rare liquid biopsy markers.
COC devices can be mass produced by injection moulding and photo-
activated using UV/O3 irradiation to yield surface-confined carboxylic
acid (-COOH) scaffolds.20 The PC linker contains a primary amine with
an ethylene glycol spacer for EDC/NHS coupling to the surface -COOH
groups, and the linker’s opposing -COOH covalently anchors Abs via
a second EDC/NHS reaction. Solid-phase conjugation prevents PC
linker cross-linking, and identical reaction chemistry simplifies
synthetic preparation and subsequent derivatization reactions.
However, EDC/NHS reactions in aqueous buffers can result in NHS
ester hydrolysis. The resulting surface -COOH groups can directly
attach Abs to the surface during the second EDC/NHS reaction. These
directly-attached Abs would not allow photorelease of the enriched
liquid biopsy marker. To mitigate NHS ester hydrolysis, we
investigated the ability to perform EDC/NHS reactions in an
anhydrous solvent, such as acetonitrile (ACN).
COC is stable in many organic solvents, but its stability is unknown
following UV/O3 activation, which generates various oxidation
products and polymer fragments.20 Surface analyses (Table S1) of
UV/O3-activated COC exposed to ACN showed increased
hydrophobicity (64.7° water contact angle vs 56.7° in buffer), ~3-fold
lower -COOH surface densities, but constant ATR-FTIR oxidation
signals. Loss of –COOH groups at the surface was presumably due to
solubilization of oxidized polymer fragments.
Figure 1. (A) Cy5-oligonucleotide reporters were immobilized via the PC linker at 0.2-5X
of the theoretical monolayer reaction excess (0.11–2.65 mM in ACN) considering the
microfluidic device’s surface area. (B) On-chip microscopy (n= 3) and (C) fluorescence
spectroscopy of released Cy5 molecules (n = 3) show saturation at 1–5X. (D) Cy5-labeled
oligonucleotides (n = 3), EVs (n=5), and SKBR3 cells (n=3) were released with 88%, 91%,
and 94% efficiency in 2 min, respectively.
The PC linker concentration required to bind the maximum
number of Abs was required to maximize recovery of the target. For
that, we varied the PC linker reaction excess compared to a
monolayer (considering surface area) and observed the same
number of Cy5-labeled reporters released in 1-5X PC linker excess
(Figure 1C). Thus, we could saturate the device at 1X PC linker
reaction excess (0.56 nmol/cm2, 1.82 x 1015 molecules per device)
and the PC linker concentration was sufficient to maximize the
recovery. We varied LED exposure time and released 74 ±6% Cy5-
labeled oligonucleotides in 1 min and 88 ±1% release efficiency in 2
min, Figure 1D, resulting in release of 6.4 ×1012 molecules (Figure 1C).
The PC linker was used to immobilize anti-EpCAM Abs in a
microfluidic device for CTC affinity-enrichment.6 EpCAM(+) SKBR3
cells were spiked into healthy donor blood (69-269 SKBR3 cells/mL)
to evaluate the assay performance with PC linker. We pre-stained
SKBR3 cells with Hoechst dye and, after enrichment, stained all cells
with SYTO 82, a membrane-permeable nuclear dye that is spectrally
distinct from Hoechst. SKBR3 cells were dual-stained, while
leukocytes were only stained with SYTO 82 enabling the
determination of recovery by a self-referencing method13 and purity
of the enriched fraction (Figure 2A).
We tested the immobilization efficiency using a 3’ Cy5-labeled
oligonucleotide reporter bearing a primary amine on its 5’ end to
UV/O3-COC surfaces pre-treated with ACN or with MES buffer (pH
4.8). ACN treatment yielded oligonucleotide reporter surface
coverages comparable to MES buffer treatment (Figure S2A).
However, improved reaction efficiency was observed when
conducting the EDC/NHS coupling reaction in ACN (Figure S2B) likely
due to eliminating NHS ester hydrolysis.
We next immobilized the PC linker (in ACN and TEA) to
UV/O3-activated COC microfluidic devices and measured the
immobilization efficiency using the Cy5-labeled oligonucleotide. TEA
2 | J. Name., 2012, 00, 1-3
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However, UV irradiation can damage nucleic acids through
DOI: 10.1039/C9CC09598E
photo-absorption and oxidation (8-oxoguanine, 8-oxo-G,
View Article Online
We next used the PC linker to immobilize anti-EpCAM Abs in a
sinusoidal microfluidic device for CTC affinity-enrichment.6 We
targeted SKBR3 cells spiked into blood (50-27069-269 SKBR3
cells/mL) to evaluate possible interferences of the sample matrix on
the PC linker’ performance. We pre-stained SKBR3 cells with Hoechst
dye and, after enrichment, stained all cells with SYTO 82, a
membrane-permeable nuclear dye that is spectrally distinct from
Hoechst. SKBR3 cells were dual-stained, while leukocytes were only
stained with SYTO 82 enabling the determination of the PC linker’s
performance metrics of recovery using a self-referencing method13
and purity of the enriched fraction (Figure 2A).
production). To assess that, we measured 8-oxo-G levels in RNA and
DNA for Hs578T cells exposed to visible LED and UV light (both at a
dose of 18.5 J) and compared to H2O2, which is known to generate
oxidative damage in DNA and RNA via oxygen free radicals. DNA
damage was not detected for LED exposure but was present with UV
irradiation (Figure 2F). Both exposures generated 8-oxo-G damage in
RNA at comparable levels to that of H2O2 (Figure 2F). Single-stranded
RNA is easily oxidized so as to protect genomic DNA from damage21
and subsequent mutations through imperfect repair pathways.22
A
gene panel consisting of mesenchymal and EMT markers were
selected to determine the impact of mRNA oxidative damage (Figure
2G; see Table S2 for primers used for the RT-qPCR). The mRNA
expression was similar in all 3 treatments suggesting that while the
8-oxo-G damage is observed in RNA, the frequency was so low that
it did not appear to alter the mRNA expression for the selected gene
panel. Collectively, visible LED exposure did not affect mRNA
expression or cause oxidative DNA damage, whereas UV irradiation
induced DNA 8-oxo-G damage. Such DNA damage could cause false
positives for clinical analysis, especially at the single cell level, which
is commonly encountered when analyzing CTCs.
We also evaluated the impact of attached Abs of enriched cells in
terms of mRNA expression following photorelease using a series of
stress genes. Affinity isolated SKBR3 cells were released, and mRNA
expression was analysed via RT-qPCR. We observed statistically
similar mRNA expression profiles (Figure S6) in both released and
cells not containing attached Abs.
Figure 2. (A) Performance of sinusoidal microfluidic device using PC linker for anti-
EpCAM enrichment of SKBR3 cells spiked into whole blood (N = 3). (B) LED release had
no effect on viability, and (C–E) released cells in culture for 2–96 h (Scale bars = 100 µm).
(F) DNA/RNA oxidative damage (N = 3) assessed for 2 min LED exposure, equivalent UV
dose (18.5 J), and 300 µM H2O2 (30 min) of Hs578T cells. DNA and RNA damage is
normalized to control. (32.2 pg 8-oxo-G per 400 ng DNA and 7.15 pg 8-oxo-G per 400 ng
RNA). The DNA-derived ELISA 8-oxo-G calibration curve could not quantify RNA damage
absolutely. (G) mRNA profiling by RT-qPCR (N = 3) of Hs578T cells following no
irradiation, LED or UV light exposure, or H2O2 treatment.
Figure 3. EVs affinity-enrichment (anti-CD8 mAbs). The EVs were enriched from
MOLT-3 conditioned media and released by LED exposure from the EV
enrichment microfluidic device. The released EVs were subjected to NTA (A), TEM
(B), and ddPCR (C). The ddPCR was carried out on 8 genes. Among them, five
genes (PLBD1, FOS, MMP9, CA4 and VCAN) are known to be dysregulated as a
result of AIS event. See Table S3 for the sequences of the primers used for the
Lastly, we tested the PC linker for EV catch and release. Our
group recently affinity enriched CD8(+) EVs as a liquid biopsy marker
to diagnose AIS. We relied on the dU linker strategy to release EVs,23
but the 60 min enzymatic reaction increased total assay time,
approaching the ~4.5 h therapeutic time window. Thus, we
investigated the use of the PC linker to reduce processing time for
releasing AIS-associated EVs. We immobilized the PC linker and anti-
CD8 Abs to a UV/O3-COC microfluidic device specifically-designed to
enrich EVs with high efficiency.23 The expression of CD8 antigen in
MOLT-3 cells was reported as 13.5%, thus the MOLT-3 cell line was
used as a model for these studies.24 CD8(+) EVs were affinity selected
from MOLT-3 conditioned media and photoreleased for NTA and
TEM analysis. From the MOLT-3 conditioned media, we enriched 8.2
±0.2 × 107 nanoparticles (NPs) with an EV size of ~136 nm, similar to
TEM (Figure 3A, B). EV release was rapid and efficient; 82 ±6% of EVs
released after 1 min LED exposure, and 91 ±5% released within 2 min
of exposure (Figure 1D). The PC linker’s rapid cleavage reduces AIS
assay workflow by >58 min compared to enzymatic release.
SKBR3 cells were enriched with 85 ±8% purity (16-38
leukocytes/mL) and 73 ±4% recovery (47-202 cells), slightly lower
than found for the dU linker (85 ±4%) and direct Ab attachment (96
±12%).13 We also observed ~40% fewer Cy5-labeled oligonucleotides
immobilized through the PC linker compared to direct conjugation to
the surface.13 We suspect that Ab load and CTC recovery may
increase if the PC linker contained a longer PEG spacer, which would
allow for more access to targets and potentially less surface crowding.
We used the PC linker to immobilize IgG 2A isotype Ab to evaluate
nonspecific cell recover, which was 3 ±2%. The release of SKBR3 cells
was rapid with 94 ±4% efficiency after 2 min of LED exposure (Figure
1D). Other breast cancer cell lines were also enriched and released
with 88 ±10% and 91 ±4% release efficiency for MCF7 and Hs578T
cells, respectively. We also found that the release efficiency was
independent of cell antigen expression (Figure S5A for flow
cytometry results and correlation to release efficiency, Figure S5B).
After exposure to visible LED light, 94 ±1% of released SKBR3 cells
were viable, the same as controls (Figure 2B), and could be
Further, the EVs mRNA gene profile was obtained by droplet
propagated in culture for 96 h (Figure 2C – E). Released MCF7 and digital PCR (see Table S3 for primer sequences) after 2 min of LED
exposure and compared with control EVs (not exposed to blue light).
Hs578T cells had 96 ±6% and 99 ±3% cell viability, respectively.
This journal is © The Royal Society of Chemistry 20xx
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The gene panel consisted of genes dysregulated as a result of AIS and 5.
stress genes (i.e., PLBD1, FOS, MMP9, CA4, VCAN and IL8). CD81 and
CD8 genes are EV-specific and used to confirm the selected EV
markers. A strong positive correlation (0.99) of the two data sets 6.
(Figure S7) implied 2 min of LED exposure did not significantly affect
EV-mRNA cargo integrity (Figure 3C).
S. R. Pullagurla, M. A. Witek, J. M. Jackson, M. A. Lindell,
M. L. Hupert, I. V. Nesterova, A. E.DBOaIi:rd10a.1n0d3V9Si/e.CwA9A.CrStCico0lep9Oe5rn9,li8nEe
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M. A. Witek, R. D. Aufforth, H. Wang, J. W. Kamande, J. M.
Jackson, S. R. Pullagurla, M. L. Hupert, J. Usary, W. Z.
Wysham, D. Hilliard, S. Montgomery, V. Bae-Jump, L. A.
Carey, P. A. Gehrig, M. I. Milowsky, C. M. Perou, J. T.
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We successfully demonstrated a PC linker for the “catch and
release” of clinically-relevant liquid biopsy markers (CTCs and EVs)
attached to a –COOH surface using two-step EDC/NHS coupling
chemistry. The PC linker is easily adaptable for any affinity agent 7.
bearing a primary amine, such as Abs and aptamers, as well as 8.
different microfluidic platforms. Elements in biologically complex
matrices, such as blood, did not interfere or affect the assay as 9.
judged by high cell recovery (>70%). We also showed high cell
viability and the possibility of culturing released CTCs. Unlike UV
exposure, our PC linker cleaves in response to visible light, and thus,
does not affect DNA integrity. Importantly, our PC linker was able to 10.
release enriched liquid biopsy markers efficiently (>90%) and rapidly
(2 min) with a blue LED (400-450 nm). AIS diagnostics takes full
advantage of the PC linker’s rapid release to reduced assay time for
diagnostic tests that possess short therapeutic time windows. This
reagent-free release method is inexpensive and well-suited for
clinical settings because it obviates the need for thermally mediated 11.
enzymatic reactions. While ambient light can cause photocleavage,
once the enrichment device has been loaded with the affinity agent
using the PC linker, the device can be wrapped in a rubylith film to 12.
protect the integrity of the PC linker (see Figure S2A).
Z. Ao, E. Parasido, S. Rawal, A. Williams, R. Schlegel, S. Liu,
C. Albanese, R. J. Cote, A. Agarwal and R. H. Datar, Lab
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E. Reategui, N. Aceto, E. J. Lim, J. P. Sullivan, A. E. Jensen,
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Conflicts of interest
There are no conflicts to declare.
13.
14.
S. V. Nair, M. A. Witek, J. M. Jackson, M. A. Lindell, S. A.
Hunsucker, T. Sapp, C. E. Perry, M. L. Hupert, V. Bae-Jump,
P. A. Gehrig, W. Z. Wysham, P. M. Armistead, P. Voorhees
and S. A. Soper, Chem. Commun., 2015, 51, 3266-3269.
J. Dong, Z. Xun, Y. Zeng, T. Yu, Y. Han, J. Chen, Y. Y. Li, G.
Yang and Y. Li, Chemistry–A European Journal, 2013, 19,
7931-7936.
Q. Huang, C. Bao, W. Ji, Q. Wang and L. Zhu, J. Mat.
Chem., 2012, 22, 18275-18282.
Q. Lin, C. Bao, S. Cheng, Y. Yang, W. Ji and L. Zhu, J Am
Chem Soc, 2012, 134, 5052-5055.
S.-W. Lv, J. Wang, M. Xie, N.-N. Lu, Z. Li, X.-W. Yan, S.-L.
Cai, P.-A. Zhang, W.-G. Dong and W.-H. Huang, Chem. Sci.,
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Q. Lin, C. Bao, S. Cheng, Y. Yang, W. Ji and L. Zhu, J. Am.
Chem. Soc., 2012, 134, 5052-5055.
R. S. Givens, M. Rubina and J. Wirz, Photochem Photobiol
Sci, 2012, 11, 472-488.
J. M. Jackson, M. A. Witek, M. L. Hupert, C. Brady, S.
Pullagurla, J. Kamande, R. D. Aufforth, C. J. Tignanelli, R. J.
Torphy, J. J. Yeh and S. A. Soper, Lab Chip, 2014, 14, 106-
117.
The authors would like to thank the NIH for funding of this work
(NIBIB: P41 EB020594; NCI: P30 CA168524, R01 CA211720; NIGMS:
P20 GM130423, P20GM103638). We also acknowledge the KU
Microscopy and Analytical Imaging Laboratory for TEM imaging
(DOD-47040-2974000-908), KU Endowment Funds (RSG). We thank
the Biospecimen Repository Core for providing healthy donor blood.
15.
16.
17.
Electronic Supporting Information (ESI)
ESI includes experimental methods: PC linker synthesis and
characterization; and immobilization; LED light exposure system; PC
release effect on DNA damage; mRNA expression analysis; and
results: synthesis of PC linker, Scheme S1; photocleavage of PC
linker, Figure S1; anhydrous solvent effects, Table S1; LED exposure
system, Figure S2; ACN vs. MES buffer EDC/NHS reactions, Figure S3;
Cy5 reporter assay, Figure S4; flow cytometry of cell lines, Figure S5;
Figure S6 gene expression of Hs578T and SKBR3 cells, Table S2;
MOLT-3 EV mRNA profile, Table S3).
18.
19.
20.
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Journal Name
COMMUNICATION
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DOI: 10.1039/C9CC09598E
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